Mitochondrial gene expression in Trypanosoma brucei is regulated at multiple posttranscriptional levels including polycistronic precursor processing, RNA editing, and RNA stability. Although these processes are critical for proper gene expression during the T. brucei life cycle, the trans-acting factors that govern their regulation are largely unknown. Pentatricopeptide motif containing proteins (PPRs) comprise a recently described family of RNA binding proteins that are particularly abundant chloroplasts and mitochondria. The few PPRs that have been functionally characterized are primarily involved in the processing, stability, and/or translation of specific organellar RNAs in plants and fungi. We identified six proteins containing multiple PPR motif repeats in the available T. brucei databases. Each of these proteins also contains a putative mitochondrial-targeting signal at its N-terminus. Our hypothesis is that the six newly identified TbPPRs modulate specific posttranscriptional gene regulatory events in T. brucei mitochondria. To test this hypothesis, we will create cell lines depleted in each of the PPRs using RNA interference and determine the effect of PPR protein knock-down on cell growth and morphology. We will then analyze the functional consequences of depleting each PPR on the processing and/or stability of specific mitochondrial RNAs. To this end, we will isolate RNA from un-induced and induced knock-down cell lines and assess 1) RNA stability using northern blots, 2) RNA editing using poisoned primer extension, and 3) precursor transcript processing by RT-PCR. Finally, we will confirm the mitochondrial localization of each of the six T. brucei PPRs and assess the potential dual localization of three of the proteins to nuclei by transfection of trypanosomes with GFP-PPR fusion proteins. These studies will provide significant new information regarding the mechanisms of mitochondrial gene regulation in trypanosomes. Since the functions of PPRs are not well understood in general, they will likely provide novel insights into the functions of this conserved protein family in higher organisms as well. [unreadable] [unreadable]